Detection and/or characterization of diseases and/or ailments in a host can be a complex process that may typically involve the identification of one or more causative agents (e.g., pathogens). There may also frequently have existed a need and/or desire to detect, characterize and/or identify one or more poisons, toxins, and/or genetic expression factors.
Substantially spherical particles (also known as microspheres or microbeads) bearing identifiable labels and/or markings—colloquially called “barcoded microbeads”—may have been used in parallel multiplexed analyses and/or in the identification of disease-related targets, toxin-related targets, and/or gene-related targets. Barcoded microbeads may have been previously conjugated to biorecognition molecules (“BRMs”)—i.e., to molecules having an affinity for, and/or an ability to interact with, one or more specific targets. Different targets may be bound to corresponding BRMs conjugated with barcoded microbeads, such as to enable identification of the targets.
Dye-labeled fluorescent microspheres may have been previously considered as a potential alternative to traditional microarrays, insofar as they may have been thought to allow for multiplexed color detection with a measure of flexibility in target selection, somewhat improved binding rates, and/or reduced costs in production. Dye-labeled fluorescent microbead-based arrays may have been thought to allow for use of different sized microbeads and/or different colored microbeads, so as to permit identification of different bead populations individually linked to specific biomolecules. The functionality of dye-labeled fluorescent microbead-based arrays may, however, have heretofore relied heavily upon one or more properties of the microspheres utilized (e.g., size, stability, uniformity, and/or ability to retain fluorescent dyes).
Previously, polymeric dye-labeled fluorescent microspheres may have been one of the most widely used microsphere systems. Polymer matrices may have advantageously protected the embedded dyes from external quenching agents, photobleaching, and/or the effects of solvent polarity, pH and/or ionic strength, possibly whilst also providing surface reactive functional groups for conjugation with different compounds, and possibly without overly negatively affecting the fluorescent properties of the microspheres.
Unfortunately, however, there has not yet been developed any simple one-step method for the large scale manufacture of labeled and/or marked polymer microbeads having a uniform shape, homogenous distribution, and/or controlled fluorescent properties.
Moreover, the use of polymer microbeads as probes in multiplexed diagnostic analyses, in which the microbeads are designed to bind to specific targets, may require not only that the various types of microbeads be detectable, but also for them to be distinguishable from one another. This kind of barcoding can be accomplished by embedding the beads with nanometer-sized fluorophores, such as quantum dots (QDs).
QDs are semiconductor nanoparticles that may exhibit size-tunable and composition-tunable fluorescence emission of symmetric and/or narrow bandwidths. QDs may typically exhibit optical and/or electronic properties that may usually be observed neither in discrete atoms, nor in bulk solids. These properties of QDs may be attributable to their physical dimensions (i.e., they are typically smaller than the exciton Bohr radius). In the result, quantum confinement may cause QDs to exhibit their somewhat unique (size-dependent) properties.
Though not essential to the working of the present invention, it may be generally thought that, with smaller and smaller QDs, the band gap energy increases, as does the energy of the photons emitted. For example, blue-light emitting QDs may be one of the smallest, if not the smallest, sized QDs which emit visible light. Conversely, the larger the size of the QD, the smaller the band gap energy. The color of the fluorescence emitted by larger QDs may, therefore, be situated generally toward the red end of the visible light spectrum.
In addition to their optical tunability, QDs may have broad excitation profiles and narrow, symmetric emission spectra. These features, among others, may make them well-suited to optical multiplexing and/or for use in association with optical barcoding technologies.
A wide variety of somewhat well-characterized QDs may be presently available. The most common may be composed of atoms from group IIB-VIB, group IIIB-VB and/or group IVB-IVB elements in the periodic table. The core of a QD may often be passivated with a cap formed from a second semiconductor which possesses a band gap energy that is greater than that of the core. For example, combinations of elements in groups IIB-VIB may sometimes be suitable second semiconductors. One commonly used QD may consist of a ZnS-capped CdSe core.
Compared to organic dyes, QDs may have similar and/or slightly lower quantum yields. This feature of QDs may be compensated for by their somewhat broader excitation profiles, higher extinction coefficients, and/or much reduced photobleaching. The size-dependent properties of QDs which might make their use preferable in comparison to dyes may also, however, be what makes them more difficult to manipulate.
The incorporation of QDs into polymer microbeads, as an alternative to organic dyes, may create additional manufacturing challenges and/or may increase the need for high quality, uniform and/or stable polymer beads.
Accordingly, it may be desirable and/or necessary to provide a method and/or system that allows for large scale manufacture of polymer microbeads. Preferably, such a system and/or method may allow for the incorporation of QDs and/or for the control of various parameters, such as the following: (i) bead diameter, (ii) degree of monodispersity, (iii) bead surface morphology, and/or (iv) rate of production—i.e., high-throughput.
One prior art approach for encapsulating QDs into preformed polystyrene microbeads may have involved swelling the microbeads in an organic solvent and in the presence of QDs. In this manner, the QDs may have been allowed to diffuse into the polymer matrices. The microbeads may then have been subsequently shrunk by evaporating the organic solvent, so as to leave the QDs ‘trapped’ inside. Major drawbacks of this prior art technique may have included difficulties in controlling the QD density inside the beads and/or diffusion of QDs out from the polymer matrices.
Other manufacturing approaches for the production of QD-doped polymeric microbeads may have previously involved, for example, batch polymerization syntheses. In such techniques, the polymerization may have taken place substantially contemporaneous with the incorporation of QDs. Problems encountered with this type of approach may have included poor control of bead diameter and/or lack of monodispersity.
Flow focusing techniques may have been previously used in making dye-labeled fluorescent polymer microspheres (A. M. Ganan-Calvo et al., International Journal of Pharmaceutics 324, (2006) 19-26). A number of U.S. patent references may also relate generally to flow focusing technologies for making dye-labeled fluorescent polymer microspheres, including the following: issued U.S. Pat. No. 6,119,953 (Ganan-Calvo), published U.S. patent application Ser. No. 10/649,376 (Ganan-Calvo), and published U.S. patent application Ser. No. 11/615,732 (Ganan-Calvo). Heretofore, however, it has not been readily apparent to those of ordinary skill in the art how one might adapt such flow focusing techniques to make polymer microbeads incorporating nanoparticles (e.g., in particular, QDs and/or magnetic nanoparticles), inter alia, in a one-step method.
A number of problems have instead presented themselves in this regard. Specifically, the flow focusing approach has thus far failed to account for certain technical considerations required to incorporate QDs into polymer microbeads—e.g., QD solubility and stability in the solvent of choice, polymer solubility and compatibility with the QD/solvent system.
One particularly problematic shortcoming of existing flow focusing technologies is their general failure to account for how QD-doped polymer microbeads might be designed for subsequent conjugation to BRMs. In this regard, the polymer of choice must not only be soluble in the QD/solvent system (and not cause the QDs to precipitate out of solution), but the chosen polymer must also have structural features which provide for finished microbeads with surfaces of the appropriate functionality—i.e., to conjugate with the BRMs. It may also be preferable to provide a one-step process for functionalizing the surfaces, so as to help eliminate any subsequent functionalization of the beads which might otherwise be required after their initial synthesis.
In the past, the surfaces of existing microbeads may have been subsequently functionalized with carboxylic acid groups, since these groups may be readily suitable to couple with the amine group of a BRM, so as to covalently bond the BRM to the surfaces of the beads. Prior art polymers with carboxylic acids in their repeating units may, however, have presented a solubility challenge, since they may have been too hydrophilic to dissolve in solvents that are compatible with QDs.
It is, therefore, an object of one preferred embodiment according to the invention to provide a method and/or a system for forming microbeads.
It is an object of one preferred embodiment according to the invention to provide a method and/or a system for forming polymer microbeads.
It is an object of one preferred embodiment according to the invention to provide a method and/or system for forming surface functionalized polymer microbeads.
It is an object of one preferred embodiment according to the invention to provide a method and/or system for forming surface functionalized barcoded polymer microbeads.
It is an object of one preferred embodiment according to the invention to provide a method and/or system for forming surface functionalized nanoparticle-doped polymer microbeads.
It is an object of one preferred embodiment according to the invention to provide a method and/or system for forming surface functionalized QD-doped polymer microbeads.
It is an object of one preferred embodiment according to the invention to provide a method and/or system for forming surface functionalized polymer microbeads that avoid and/or overcome one or more problems previously associated with the large scale manufacture of polymer microbeads.
It is an object of one preferred embodiment according to the invention to develop a one-step method and/or system for the large scale manufacture of barcoded polymer microbeads having a uniform shape, homogenous distribution and/or controlled and readily identiable properties.
It is an object of the present invention to obviate or mitigate one or more of the aforementioned disadvantages associated with the prior art, and/or to achieve one or more of the aforementioned objects of the invention.